半导体量子点-Smad4单克隆抗体荧光探针的制备及免疫特异识别能力的检测

Preparing of semiconductor quantmn dots-Smad4 monoclonal antibody fluorescent probes and identification of their ability of specific immuno-recognition

目的制备半导体量子点（semiconductor quantumdots，SQD）单克隆抗体荧光探针-Smad4（SQD-Smad4），并检测其光学性质和免疫特异性识别能力，为视踪检测活细胞内蛋白质等生物分子提供实验手段。方法用化学偶联法制备水溶性SQD-Smad4探针，测定其荧光比值、光稳定性、吸收光谱和发射光谱；采用SP免疫组化法和SQD-Smad4直接免疫荧光成像法观察比较Smad4在大鼠牙乳头细胞内的分布。结果在紫外线持续照射12h前后605SQD和605SQD-Smad4的相对荧光比值分别为250、254和240、242。SQD与Smad4单抗通过共价结合形成稳定的SQD．Smad4，其对大鼠牙乳头细胞内Smad4分子在TGF-β1刺激牙乳头细胞24h后发生核移位，说明具有特异性的识别能力；SQD-Smad4仍具有SQD所具有的激发光谱宽、发射光谱窄、荧光度强、光化学稳定性好等光学特征。结论SQD和单抗共价结合形成分子探针后仍具有特异免疫识别能力和独特的光学性质，为SQD原位、长时间视踪检测活细胞内生物分子运动提供了实验基础。

Objective To prepare semiconductor quantum dots （SQD）-Smad4 monoclonal antibodyfluorescent probes and to detect the optical qualities and the ability of specific recognition of the probes. Methods SQD were chemically modified with Smad4 monoclonal antibody proteins to prepare water soluble probes, and the fluorescence intensity, photostability, absorption spectra and emission spectra of the probes were studied. The location of Smad4 in rat dental papillae cells （RDPC） was examined by SP anti- Smad4 immunocytochemical method and SQD-Smad4 direct immunofluorescent imaging. Results SQD and monoclonal antibody covalently bonded to form the fluorescent probes which could specifically recognize Smad4 in RDPC. These fluorescent probes still had properties, including broad absorption band, narrow emission band, high fluorescence intensity and photostability. Conclusions SQD and monoclonal antibody could covalently bond to form the fluorescent probes with distinct optics character and ability of specific recognization,which provides the scientific evidence that SQD trace the molecular movement in living cells in long-term, in situ and in real time.